Type III secretion is a method of delivering toxic proteins directly into host cells via a molecular syringe- like structure, which is employed by many Gram-negative pathogens. Type III secretion systems contribute to an incredibly diverse range of diseases in humans including bubonic plague, food-borne illnesses, and cystic fibrosis. The millions of cases each year of food-borne illnesses alone have a staggering impact on our health care system. Add to that the increasingly deadly potential from emerging antibiotic resistance, and the need for new antimicrobial treatments is urgent. Though the toxins that are delivered by individual pathogens vary, the fundamentals of assembling and deploying type III secretion systems are well conserved and represent a potential therapeutic target. To design effective strategies that disable this virulence factor, a better understanding of its architecture and regulation is necessary. Type III secretion systems are generally only made and activated during infection of a susceptible host. During infection, bacteria assemble the needle-like structure, but toxins are not secreted through it until contact with a host cell is made. How do bacteria activate the secretion system upon cell contact? Just as important, how do they shut if off? These questions are fundamental to our understanding of the system, and these questions are the focus of this proposal. We study the Yersinia type III secretion system because it is a well-established model system. Our novel tools and approaches have revealed new insight into the heart of type III secretion regulation. In particular, we have found that YopK, a regulator of type III secretion, has two distinct regulatory functions and works at both ends of the needle. I appears that YopK is an integral component of an ON/OFF switch for secretion. From the bacterial side, YopK helps to monitor which proteins are secreted through the needle. YopK itself is secreted and once inside the host cell, YopK interacts with the end of the needle complex to shut down secretion of the toxins. In this proposal we will investigate how YopK performs these activities. Using a combination of genetics, cell biology, and biochemistry we will determine how YopK coordinates with other known secretion regulators on both sides of the needle. We will also identify and characterize the functional domains within YopK that mediate its activities. Collectively, our work will reveal fundamental mechanisms controlling this important virulence weapon. If we understand the nature of the ON/OFF switch, we can design a new type of broad-spectrum drug that targets a vital secretion system in a wide range of bacteria.